Novel tricarbocyanine-cyclodextrin(s) conjugates and use thereof

ABSTRACT

The present invention relates to novel tricarbocyanine-cyclodextrin(s) conjugates useful as markers in the diagnosis of kidney diseases, a diagnostic composition comprising said conjugates, their use and their production.

FIELD OF THE INVENTION

The present invention relates to novel tricarbocyanine-cyclodextrin(s)conjugates, and use thereof as diagnostic agents for kidney diseases.

BACKGROUND OF THE INVENTION

Fructans are used as markers in kidney diagnostics and in particular todetermine the glomerular filtration rate (GFR) as a test for kidneyfunction.

Fructans are straight or branched chain oligosaccharides andpolysaccharides with an sucrose terminal end. Fructans can havedifferent physical properties, such as water solubility depending on thedegree of branching and polymerization. Fructans occur in plants ascarbohydrate reserves. As a natural product the fructans have anunpredictable length.

The fructans inulin and sinistrin are used in particular as markers inkidney function tests. Inulin and sinistrin are composed of 10 to 40fructose units with a corresponding molecular weight of 1600 to 6500 Da.After intravenous injection, inulin and sinistrin are neither changednor stored in the organism, but they are filtered out by the kidneyglomeruli and are not reabsorbed again in the tubuli. The filtration ofthe fructans may vary according to their size.

In order to assess kidney function, it is usual to determine the timevariation of the concentration of the marker in the blood afterintravenous injection of said marker. To do so, blood samples have to bedrawn. The concentration marker in the blood may, for example, bedetermined by enzymatic methods, as described in Kuehnle et al.,Nephron, 62, 104-107 (1992). This method is time consuming, verycumbersome and of limited use.

A simpler alternative, based on fluorescein isothiocyanate labelledinulin (FITC-inulin) was described (M. Sohtell et al., Acta Physiol.Scand 119, 313-316 (1983); J. N. Lorenz and E. Gruenstein, Am. J.Physiol. (Renal Physiol. 45) 276, F172-F177 (1999). With this approach,also, blood is sampled and the fluorescein label of the FITC-Inulin isdetermined in the plasma. A very serious problem with this approach isthat hemolysis, occurring during blood sampling, affects thedetermination of the concentration. Moreover, hemoglobin absorbs theexcitation light at 480 nm very well, so less light is emitted and theapparent concentration is lower than in reality.

A disadvantage of inulin and FITC-inulin for the clinical routineanalyses is their very low solubility in water. Hence the preparationscontaining insulin and FITC-insulin have to be heated to 90° C. untilcomplete dissolution (Rieg, T. A High-throughput Method for Measurementof Glomerular Filtration Rate in Conscious Mice. J. Vis. Exp. (75),e50330, doi:10.3791/50330 (2013)), as their aqueous solutions tend tocrystallize during storage. Unfortunately, this causes a partialdegradation of inulin to fructose. Furthermore, the solution has to bethen dialysed for 24 hr at room temperature. This step is especiallyimportant to FITC-inulin in order to remove unconjugated FITC, but alsothe byproducts generated by the heating procedure. Dialysissubstantially decreases the concentration of FITC-inulin. In addition,the low solubility of inulin and FITC-inulin makes it difficult toachieve a well defined concentration and to handle the marker during theinjection.

Recently, a Cy5.5-inulin conjugate has been introduced by Perkin-Elmer(GFR-Vivo; application note by Peterson, J. D, Perkin-ElmerCorporation). An advantage of Cy5.5-inulin conjugate over FITC-inulin isrelated to the excitation/emission wavelengths of Cy5.5 (675/705 nm).The longer wavelength of Cy5.5 allows a deeper tissue penetration, butits use with a small animal imager requires the animals beinganesthetized. Anesthesia, however, has an unpredictable impact on bloodpressure (initial rise, decrease during the major phase of anesthesia,followed by a rise at the ending phase). Kidney perfusion and thus GFRare highly sensitive to blood pressure, with low blood pressure valuesresulting in low GFR values. Thus, a meaningful/reproducible GFRmeasurement is not possible under anesthesia.

A substantial improvement over the previous art was the introduction ofa FITC-sinistrin conjugate as described in U.S. Pat. No. 6,995,019. Thismarker is much more water soluble than FITC-inulin and, in addition, noundesired circulatory reactions have been observed when usingFITC-sinistrin. Furthermore its concentration change over time can bemeasured transcutaneously. There is, however, still the problem of thepenetration depth into the skin at a given wave length (480 nm allow adepth of only a few mm) and thus the dosage of the marker has to befairly high to obtain a clear (measurement) signal. Another problem ofthe transcutaneous measurement is skin pigmentation, as melanin in theskin absorbs light quite efficiently at 480 nm.

A major disadvantage of inulin and also sinistrin is that they arenatural products; their composition is quite variable even within thesame batch, and even more in different batches. For Regulatory Affairsthis is not acceptable.

OBJECT AND SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel substancewhich can be used as marker in a kidney function test which overcome thedisadvantages of the markers known in the prior art.

According to the invention, the above object is achieved thanks to thematter specified in the ensuing claims, which are understood as formingan integral part of the present invention.

The invention relates to fluorescent tricarbocyanine-cyclodextrin(s)conjugates as markers for kidney function tests in mammals.

An embodiment of the present invention relates to a fluorescent compoundof formula (I)

F-L_(n)-CD_(n)  (I)

wherein

F is a tricarbocyanine residue of formula (II)

wherein

R₁ and R₂ are independently selected from H, SO₃H, CO₂H, SO₂NH₂,CH₂COOH, NH₂, NHCOCH₂I, NO₂, Br, Cl, CH₃;

R₃ and R₄ are independently selected from C₁₋₄ alkyl, (CH₂)₃C≡CH,(CH₂)₄C≡CH (CH₂)₅COOH, (CH₂)₃SO₃H, (CH₂)₄SO₃H, (CH₂)₃NH₂, (CH₂)₄NH₂,(CH₂)₃N⁺(CH₃)₃, (CH₂)₅N⁺(CH₃)₃, (CH₂)₃N₃, (CH₂)₄N₃, (CH₂)₃NHCOCH₂I,(CH₂)₄NHCOCH₂I; (CH₂CH₂O)₂CH₃, (CH₂CH₂O)₃CH₃ (CH₂CH₂O)₄CH₃;

R₅, is H, Cl, or

wherein

X is selected from NH, O, S;

j is an integer from 1 to 4;

k is an integer from 1 to 4;

CD is a cyclodextrin residue of formula (III)

wherein

m is and integer equal to 6, 7 or 8,

R′, R″, R′″ are independently selected from OH, OCH₃, OCH₂CH₃,OCH₂CHOHCH₃, OCHOHCH₃, OCH₂COOH, O(CH₂)₄SO₃H, N₃, NH₂, NHCOCH₃,OCH₂C≡CH, SH;

L is a linker group resulting from the coupling of the tricarbocyanineof formula (II) to the cyclodextrin(s) of formula (III) according to thefollowing Table 1:

Functional group of Functional group the tricarbocyanine of thecyclodextrin (F) in any (CD) in any of the groups of the groups R₃, R₄or R₅ R′, R″ or R′′′ Linker group (L) COOH OH —C(O)O— COOH NH₂ —C(O)NH—NCS OH —NC(S)O— NCS NH₂ —NC(S)NH— NH₂ COOH —NHC(O)— NHCOCH₂I SHNHC(O)CH₂S— C≡CH N₃

N₃ C≡CH

dichlorotriazine OH

dichlorotriazine OH, OH

dichlorotriazine OH, NH₂

n is an integer from 1 to 4,

and salts thereof.

A further embodiment of the present invention relates to a diagnosticformulation comprising at least one fluorescent compound of formula (I)for use in diagnostic tests for determining the kidney functionparameters, preferably the glomerular filtration rate (GFR), of amammal.

A still further embodiment of the present invention relates to adiagnostic method for determining whether a mammal suffers from chronickidney diseases, wherein the method comprises i) administering at leastone fluorescent compound of formula (I) or a diagnostic formulationcomprising at least one compound of formula (I) to the mammal, and ii)detecting and measuring the fluorescence emitted by the fluorescentcompound, wherein the measured fluorescence directly correlates (i.e.correlates in a proportional way) with the kidney function, particularlythe glomerular filtration rate (GFR), of the mammal.

A further embodiment of the present invention relates to a method forscreening pharmaceutical compounds suitable for treatment of chronickidney diseases, wherein the method comprises:

i) administering to an animal model of chronic kidney disease apharmaceutical compound and at least one fluorescent compound of formula(I), wherein the fluorescent compound is administered subsequently tothe pharmaceutical compound;

ii) measuring the glomerular filtration rate by detecting and measuringthe fluorescence emission of the at least one fluorescent compound offormula (I), wherein the detection and the measurement of thefluorescence comprises the detection and the measurement of thefluorescent emission emerging from the skin of the animal model inresponse to excitation with a red light or near infrared light source;and

iii) selecting the pharmaceutical compound that increases glomerularfiltration rate (GFR).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example only, withreference to the enclosed figures of drawing, wherein:

FIG. 1: Structural formula of 2-hydroxypropyl-β-cyclodextrin (HβCD).

FIG. 2: Absorption spectrum of ABZWCY-HβCD in methanol.

FIG. 3: Emission spectrum of ABZWCY-HβCD in methanol.

FIG. 4: Schematic diagram of the transcutaneous measuring device.

FIG. 5: Application to a rat of the transcutaneous measuring device.

FIG. 6: Plasma clearance kinetics of ABZWCY-HβCD: (a) 1 exponentialfitting (1e); 3 exponential fitting (3e).

FIG. 7: Plasma clearance kinetics of ABZWCY-HβCD in the presence ofProbenecid: (a) 1 exponential fitting (1e); 3 exponential fitting (3e).

DETAILED DESCRIPTION OF THE INVENTION

In the following description, numerous specific details are given toprovide a thorough understanding of embodiments. The embodiments can bepracticed without one or more of the specific details, or with othermethods, components, materials, etc. In other instances, well-knownstructures, materials, or operations are not shown or described indetail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearances of the phrases “in oneembodiment” or “in an embodiment” in various places throughout thisspecification are not necessarily all referring to the same embodiment.Furthermore, the particular features, structures, or characteristics maybe combined in any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do notinterpret the scope or meaning of the embodiments.

The invention relates to fluorescent tricarbocyanine-cyclodextrin(s)conjugates as markers for kidney function tests.

These fluorescent conjugates, that are the object of the presentinvention, are represented by the general formula (I)

F-L_(n)-CD_(n)  (I)

wherein

F is a tricarbocyanine residue of formula (II)

wherein

R₁ and R₂ are independently selected from H, SO₃H, CO₂H, SO₂NH₂,CH₂COOH, NH₂, NHCOCH₂I, NO₂, Br, Cl, CH₃;

R₃ and R₄ are independently selected from C₁₋₄ alkyl, (CH₂)₃C≡CH,(CH₂)₄C≡CH (CH₂)₅COOH, (CH₂)₃SO₃H, (CH₂)₄SO₃H, (CH₂)₃NH₂, (CH₂)₄NH₂,(CH₂)₃N⁺(CH₃)₃, (CH₂)₅N⁺(CH₃)₃, (CH₂)₃N₃, (CH₂)₄N₃, (CH₂)₃NHCOCH₂I,(CH₂)₄NHCOCH₂I; (CH₂CH₂O)₂CH₃, (CH₂CH₂O)₃CH₃ (CH₂CH₂O)₄CH₃;

R₅, is H, Cl, or

wherein

X is selected from NH, O, S;

j is an integer from 1 to 4;

k is an integer from 1 to 4;

CD is a cyclodextrin residue of formula (III)

wherein

m is and integer equal to 6, 7 or 8,

R′, R″, R′″ are independently selected from OH, OCH₃, OCH₂CH₃,OCH₂CHOHCH₃, OCHOHCH₃, OCH₂COOH, O(CH₂)₄SO₃H, N₃, NH₂, NHCOCH₃,OCH₂C≡CH, SH;

L is a linker group resulting from the coupling of the tricarbocyanineof formula (II) to the cyclodextrin(s) of formula (III) according to thefollowing Table 1:

Functional group of Functional group the tricarbocyanine of thecyclodextrin (F) in any (CD) in any of the groups of the groups R₃, R₄or R₅ R′, R″ or R′′′ Linker group (L) COOH OH —C(O)O— COOH NH₂ —C(O)NH—NCS OH —NC(S)O— NCS NH₂ —NC(S)NH— NH₂ COOH —NHC(O)— NHCOCH₂I SHNHC(O)CH₂S— C≡CH N₃

N₃ C≡CH

dichlorotriazine OH

dichlorotriazine OH, OH

dichlorotriazine OH, NH₂

n is an integer from 1 to 4,

and salts thereof.

Dyes belonging to the class of cyanines have already found some use inclinical diagnostics; in particular, Indocyanine Green has been used forkidney function test and fluorescence angiography for more than 30years.

Tricarbocyanine dyes absorb and emit light in the near-infrared region(NIR) (650-900 nm). Tricarbocyanine dyes are especially suitable for invivo imaging, diagnostics and even therapeutics, since biologicaltissues are relatively poor absorbers in the near-infrared spectralregion, and infrared light can penetrate deeply in such tissues; inaddition, these dyes do not give origin (or at very low amount) toauto-fluorescence in the near-infrared spectral region.

Cyclodextrins (CD) are cyclic oligosaccharides produced by the enzymaticdegradation of starch. Depending on reaction conditions, three main CDscan be obtained α, β and γ; they consist of 6, 7 or 8 glucopyranoseunits. They are shaped as a truncated cone, with hydroxyl groups on eachside. Their cavity is constituted by the glucosidal moieties. Thesethree dimensional structures result in a high external hydrophilicityand internal hydrophobicity.

In one embodiment, CDs are selected from β- and γ-cyclodextrins, thatis, CD in which m parameter of formula (III) has a value about of 7(β-cyclodextrin) or 8 (γ-cyclodextrin), respectively.

While the water solubility of natural CDs is limited, chemicalsubstitution at the 2-, 3- and 6-hydroxyl sites (i.e., when R′, R″ andR′″ are OH) greatly increases solubility. Most of cyclodextrins modifiedin this way, are able to achieve a 50% (w/v) concentration in water.

In one embodiment, at least one of the groups R′, R″, R′″ of formula(III) is selected from OCH₃, OCH₂CH₃, OCH₂CHOHCH₃, provided that atleast one group R′, R″, R′″ is OH.

In a preferred embodiment, R′ and R″ are OH, R′″ is selected from OCH₃,OCH₂CH₃, OCH₂CHOHCH₃, preferably is OCH₂CHOHCH₃, in which thesubstitution degree of R′″ is between 0.5 and 1.5 per unit of formula(III).

It is known to the skilled technician that the substitution of thegroups R′, R″, R′″ of a cyclodextrin molecule, for example with groupsOCH₃, OCH₂CH₃, OCH₂CHOHCH₃, is partial, i.e. it that takes place only insome of the m structures of formula (III) constituting the cyclodextrinwith a substitution degree between 0.5 and 1.5 substituents per unit offormula (III); in other words, some groups R′, R″, R′″ in some of the mstructures of formula (III) are not replaced, or are OH groups, and arethe ones predominantly, though not exclusively, involved in theconjugation with tricarbocyanine molecules of formula (II).

In a further preferred embodiment, cyclodextrins of formula (III) areselected from 2-hydroxypropyl cyclodextrins (HCD), and in particular2-hydroxypropyl-3-cyclodextrin (HβCD, whose chemical structure is shownin FIG. 2, and 2-hydroxypropyl-γ-cyclodextrin (HγCD).

HβCD and HγCD have been found to be non-toxic in mice and rabbits[Pitha, J. “Amorphous water soluble derivatives of cyclodextrins: nontoxic dissolution enhancing excipients.” J. Pharm. Sci. 1985, 74, 987].HβCD and HγCD are widely used to improve the water solubility of drugs.

HβCD and HγCD represent advantageous substitutes of fructans, such asinulin and sinistrin, as components of fluorescent markers for thedetermination of GFR, as they are relatively inexpensive, non-toxic,structurally well-defined, synthetic products, with a strongsolubilizing power.

In an embodiment, the conjugate of formula (I) presents from 1 to 4linker groups L to allow conjugation of 1 to 4 cyclodextrin molecules toone tricarbocyanine molecule.

In a preferred embodiment, R₁ and R₂ groups are independently selectedfrom H, SO₃H, CO₂H.

In a preferred embodiment, R₃ and R₄ groups are independently selectedfrom methyl, ethyl, (CH₂)₅COOH, (CH₂)₄SO₃H, (CH₂)₃N⁺(CH₃)₃.

In a preferred embodiment, R₅ group is selected from H, Cl, or

In a preferred embodiment, the linker group L is selected from an ester,an ether, an amide, a thiocarbamate, a thiourea, a thioether, a1,2,3-triazole, or

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (IV):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula (I)(corresponding to the value of the n parameter of formula (I) equalto 1) is an ester bond formed in the coupling reaction of the carboxylgroup of the tricarbocyanine (corresponding to carboxyl group of R₅group=

with a residue group R′″═OH of CD.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (V):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula (I)(corresponding to the value of the n parameter of formula (I) equalto 1) is an ester bond formed in the coupling reaction of the carboxylgroup of the tricarbocyanine (corresponding to carboxyl group of R₅group=

with a residue group R′″═OH of CD.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (VI):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula (I)(corresponding to the value of the n parameter of formula (I) equalto 1) is an ester bond formed in the coupling reaction of the carboxylgroup of the tricarbocyanine (corresponding to the carboxyl group of R₅group=

with a residue group R′″═OH of CD.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (VII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ether bonds formed in the coupling reaction of theR₅ group=dichlorotriazine of the tricarbocyanine with a residue groupR′″═OH of CD₁ and CD₂, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (VIII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl group of the R₃ and R₄ groups=(CH₂)₅COOH) with a residuegroup R′″═OH of CD₁ and CD₂, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (IX):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl group of the R₃ and R₄ groups=(CH₂)₅COOH), with a residuegroup R′″═OH of CD₁ and CD₂, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (X):

wherein CD₁, CD₂ and CD₃ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) (corresponding to the value ofthe n parameter of formula (I) equal to 3) are ester bonds formed in therespective coupling reaction of a carboxyl group of the tricarbocyanine(corresponding to the carboxyl group of the R₃ and R₄ groups=(CH₂)₅COOHand the carboxyl group of the

with a residue group R′″═OH of CD₁, CD₂, and CD₃, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XI):

wherein CD₁, CD₂ and CD₃ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) (corresponding to the value ofthe n parameter of formula (I) equal to 3) are ester bonds formed in therespective coupling reaction of a carboxyl group of the tricarbocyanine(corresponding to the carboxyl group of the R₃ and R₄ groups=(CH₂)₅COOH)and the carboxyl group of

with a residue group R′″═OH of CD₁ CD₂ and CD₃. respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl group of the R₃ and R₄ groups (CH₂)₅COOH) with a residuegroup R′″═OH of CD₁ and CD₂, respectively

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XIII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl group of the

with a residue group R′″═OH of CD₁ and CD₂, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XIV):

wherein CD₁, CD₂, CD₃ and CD₄ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) (corresponding to the value ofthe n parameter of formula (I) equal to 4) are ester bonds formed in therespective coupling reaction of a carboxyl group of the tricarbocyanine(corresponding to the carboxyl group of the

with a residue group R′″═OH of CD₁ and CD₂, CD₃ and CD₄, respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XV):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl groups of the

with a residue group R′″═OH of CD₁ and CD₂, respectively

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XVI):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) (corresponding to the value of the n parameter of formula(I) equal to 2) are ester bonds formed in the respective couplingreaction of a carboxyl group of the tricarbocyanine (corresponding tothe carboxyl groups of the

with a residue group R′″═OH of, CD₁ and CD₂. respectively.

In a preferred embodiment, the fluorescent tricarbocyanine-cyclodextrinconjugate is a compound of formula (XVII):

wherein CD is a 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula (I)(corresponding to the value of the n parameter of formula (I) equalto 1) is an ester bond formed in the respective coupling reaction of acarboxyl group of the tricarbocyanine (corresponding to the carboxylgroup of the

with a residue group R′″═OH of CD.

In a further embodiment, the present invention relates to the use of atleast one fluorescent compound of formula (I) or a diagnosticformulation comprising at least one fluorescent compound of formula (I)in kidney diagnostics, preferably in measuring the glomerular filtrationrate (GFR), in a mammal.

In an embodiment, the mammal is a mouse, a rat, a guinea pig, a cat, adog, a sheep, a goat, a pig, a cow, a horse, a primate.

In a still further embodiment, the present disclosure relates to amethod of diagnosing the glomerular filtration rate of a mammal,preferably the glomerular filtration rate (GFR), wherein at least onecompound of formula (I) or a diagnostic formulation comprising at last acompound of formula (I) is administered to a mammal and the fluorescentsignal emitted from the dye of compound of formula (I) is detected andmeasured.

Preferably, the at least one compound of formula (I) or the diagnosticformulation comprising at least one compound of formula (I) isadministered to the mammal via a parenteral route.

The diagnostic method herein disclosed is non-invasive, because thedetection and measurement of the fluorescence emitted from the at leastone compound of formula (I) are realized by detecting and measuring thefluorescent emission emerging from the skin of the mammal in response toexcitation with a red light or near infrared light source, preferably bymeans of a sensor device placed onto the mammal skin. The method isaccomplished in a clinically relevant period of time. That is, thatperiod of time is such to allow the absorption of the compound offormula (I) in the blood of the mammal and the following secretion bythe kidney system.

In a further embodiment, a compound of formula (I) can be used forscreening pharmaceutical compounds (test agents) suitable for treatmentof chronic kidney diseases.

The screening method comprises:

i) administering to an animal model of chronic kidney diseases the testagent and at least one fluorescent compound of formula (I), wherein thefluorescent compound is administered subsequently to test agent;

ii) measuring the glomerular filtration rate by detecting and measuringthe fluorescence emission of the at least one fluorescent compound,wherein the detection and the measurement of the fluorescence comprisesthe detection and the measurement of the fluorescent emission emergingfrom the skin of the animal model in response to excitation with a redlight or near infrared light source;

iii) selecting the test agent that increases glomerular filtration rate.

Mammalian models of chronic kidney diseases in, for example, mice, rats,guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and primates,may be created by causing an appropriate direct or indirect injury tothe kidney tissue of the animal. Animal models of acute kidney failuremay, for example, be created by inducing in the animal the conditions ordiseases such as acute interstitial nephritis or acute tubular necrosis,for example by the controlled administration of nephrotoxic agents(e.g., antibiotics, aminoglycoside drugs, heavy metals).

Other mammalian models of chronic kidney disease are disclosed inVukicevic, et al. (1987), J. Bone Mineral Res. 2:533; and EP-B-0 914146; animal models have been described in detail in a handbookExperimental and genetic rat models of chronic renal failure, Gretz, N.;Strauch, M.; Karger (Basel and New York); 1993; pp 343; (ISBN3805554990). Rat models of autosomal dominant polycystic kidney diseasehave been described in Gretz N, Kränzlin B, Pey R, Schieren G, Bach J,Obermüller N, Ceccherini I, Klöting I, Rohmeiss P, Bachmann S, Hafner M.Nephrol Dial Transplant. 1996; 11 Suppl 6:46-51. Review; murine modelsof polycystic kidney disease have been described in Schieren G, Pey R,Bach J, Hafner M, Gretz N. Nephrol Dial Transplant. 1996; 11 Suppl6:38-45. Review.

The following examples are intended to illustrate particular aspects ofthe present invention and should not be construed as limiting the scopethereof as defined by the claims.

The following examples provide a detailed description of the synthesisof the tricarbocyanine named2-((E)-2-((E)-2-((4-(2-carboxyethyl)phenyl)amino)-3-((E)-2-(3,3-dimethyl-5-sulfonate-1-(3-trimethylammonium)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonium)propyl)-3H-indol-1-ium-5-sulfonatebromide (named ABZWCY), its conjugation to a cyclodextrin molecule andits use in a diagnostic method for determining GFR in a rat.

A fluorescent tricarbocyanine dye ABZWCY (4) was synthesized and linkedto HβCD, according to Example 1 and Reaction Schemes 1-4.

The resulting ABZWCY-HβCD conjugate exhibited excellent water solubilitywith concentrations reaching more than 100 mg/mL. In addition it showeda low plasma protein binding (PPB), i.e. less than 10%, which is a lowervalue than ¹²⁵I-iothalamate, one of the golden standard agent for theGFR measurement [Levey, A. S. et al. J. Am. Soc. Nephrol. 1993 4(5),1159-1177].

The noninvasive real-time monitoring of plasma clearance resulted in ahalf-life of approximately 17±2 min. Moreover, thetricarbocyanine-cyclodextrin marker did not exhibit significantdifferences in plasma clearance half time in the absence and presence ofa compound able to inhibit tubular secretion. This means that kidneytubular secretion is not a significant elimination pathway for thesemarkers in a mammal. The present marker was exclusively cleared by thekidneys, with no appreciable nonspecific background signal in all thetissues and organs and only fluorescence signal remaining in the bladder2 h post injection. In conclusion, such a fluorescent compound is highlysuitable as exogenous fluorescent tracer for monitoring GFR.

Example 1 Preparation of ABZWCY-HβCD a) Preparation of2,3,3-Trimethyl-1-[3-(trimethylammonium)propyl]-3H-indolinium sulfonatebromide (1)

The reaction was carried out according to Scheme 1.

A mixture of 2,3,3-trimethyl-3H-indole-5-sulfonic acid (1.54 g, 6.5mmol; (prepared according to Mujumdar et al., Bioconjugate Chemistry(1993), 4/2, 106) and (3-bromopropyl)trimethyl ammonium bromide (2.51 g,9.5 mmol) in 1,2-dichlorobenzene (16 mL) was heated at 130° C. for 72hours under argon flow. The reaction mixture was cooled to roomtemperature and the solvent was decanted. The crude product was washedwith CH₂Cl₂, dissolved in acetone and reprecipitated into a large volumeof ethyl acetate to afford a solid 1, which was used in the next stepwithout further purification.

b) Preparation of2-((E)-2-((E)-2-chloro-3-((E)-2-(3,3′-dimethyl-5-sulfonate-1-(3-(trimethylammonium)propyl)-indo-lin-2-ylidene)cyclohex-1-enyl)-3,3-dimethyl-1-(3-(trimethylammonium)-propyl)-3H-indolium-5-sulfonatebromide (3)

The reaction was carried out according to Scheme 2.

A mixture of bromide salt 1 (0.5 g, 1.48 mmol), Vilsmeier-Haack reagent2 (0.265 g, 0.73 mmol; prepared according to Makin S. M.; Boiko, L. I.;Shavrigina, O. A. Zh. Org. Khim. 1977, 13, 1189) and anhydrous sodiumacetate (0.246 g, 3 mmol) was refluxed in 10 ml of absolute ethanol for6 h under argon flow. The reaction mixture was cooled to roomtemperature, and then concentrated under reduced pressure to yield abrown-green residue. The crude product was washed with dichloromethaneand the residue was suspended in methanol/dichloromethane (1/4, 100 mL),filtered and dried in vacuo to yield a golden-green solid (3) 505 mg,yield=84.9%.

¹HNMR (400 MHz, DMSO-d₆) δ 1.72 (s, 12H), 1.88 (m, 2H), 2.18 (m, 4H),2.76 (m, 4H), 3.08 (s, 18H), 3.49 (m, 4H), 4.18 (m, 4H) m 6.36 (d), 7.45(d) 7.85 (s, 2H), 8.31 (d, 2H). Absorption max (MeOH) 777 nm (methanol).Emission max (MeOH): 810 nm.

c) Preparation of2-((E)-2-((E)-2-((4-(2-carboxyethyl)phenyl)amino)-3-((E)-2-(3,3-dimethyl-5-sulfonate-1-(3-trimethylammonium)propyl)indolin-2-ylidene)ethylidene)-cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonium)propyl)-3H-indol-1-ium-5-sulfonatebromide (ABZWCY, 4)

The reaction was carried out according to Scheme 3.

A mixture of 3 (220 mg, 0.27 mmol) and 3-(4-aminophenyl)propanoic acid(178 mg, 1.08 mmol) in DMSO was heated at 65° C. overnight. The reactionmixture was cooled to room temperature and precipitated indichloromethane. The crude product was purified by RP C18 chromatographyto yield a blue solid 130 mg.

FW for C₅₁H₆₈N₅O₈S₂Br: 1023.15; exact mass of cation 942.45.

d) Preparation of ABZWCY-HβCD (5)

Materials:

2-hydroxypropyl-β-cyclodextrin (HβCD) was purchased from Sigma-Aldrich(Product No. H-107 of molecular formula (C₆H₉O₅)₇(C₃H₇O)_(4.5); averagemolecular weight; 1396 (anhyd.) water solubility 45 g/100 mL).

The reaction was carried out according to Scheme 4.

A mixture of dye 4 (40 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20 mg), 4-dimethylaminopyridine (10 mg) and(2-hydroxypropyl)-β-cyclodextrin (550 mg) and DMSO (6 mL) was stirredunder room temperature for 12 hours. The reaction mixture was thenprecipitated in dichloromethane. The obtained crude product was furtherpurified by Sephadex G-10 gel filtration to yield blue solid (5), 350mg.

MS (ESI) m/z=cluster of peaks between 2200-2470, average 2335; estimatedaverage m/z for ABZWCY-HβCD: 2338 (943 (ABZWCY)+1396 (HβCD, as indicatedby producer).

Absorption max=700 nm; Emission max=791 nm. Absorption and emissionspectra are shown in FIGS. 3 and 4.

Water solubility >100 mg/mL.

Example 2 Plasma protein binding (PPB) for ABZWCY-HβCD

Stock Solution Preparation.

An ABZWCY-HβCD/plasma stock solution was prepared by incubation of 500μg/ml ABZWCY-HβCD (in PBS solution) with rat plasma protein at 37° C.for 1 hour.

Equilibrium Dialysis.

Plasma protein binding measurements were performed by equilibriumdialysis technique using a two-chamber dialysis set up. 400 μl of thestock solution (see above) was placed into one side of two-chamberdialysis apparatus, another side was filled with 400 μl distilled water,and the marker-protein solution was dialyzed. At 18 and 36 hours, theconcentration of the free marker in the water side and plasma side ofthe cell was determined by absorption spectroscopy and calculated on thebasis of Beer's law.

Calculations:

PPB %=C _(marker bound to plasma) /C _(marker dye)×100

PPB %=(C _(P) −C _(W))/(C _(P) +C _(W))×100

PPB %=(A _(P) −A _(W))/(A _(P) +A _(W))×100

whereinA_(P) represents the absorbance in the plasma side of the cell afterdialysis,A_(W) represents the absorbance in water side of the cell afterdialysis.

PPB % was calculated to be 8.4% (average of 3 experiments).

Example 3 Plasma Clearance Half-Life for ABZWCY-HβCD

Plasma clearance half-life was analysed in combination with anelectronic near infrared device for the transcutaneous fluorescencedetection in rat models.

Animal:

female Sprague-Dawley rats.

Substance:

ABZWCY-HβCD Dosage:

ABZWCY-HβCD: 5 mg/100 g body weight Electronic Near Infrared Device forTranscutaneous Fluorescence Detection.

This device (sensor plaster), described in detail in US2011230739A1,“Transcutaneous Organ Function Measurement”, consists of (a) a plasterwhich can be stuck onto the skin surface; (b) a near infrared emittingdiode; (c) a radiation detector. The adhesive surface of the sensorplaster laterally encloses the detector to prevent ambient light frombeing able to pass to the detector. The near infrared radiation (peak atabout 680 nm) is partially absorbed by the marker; the responseradiation is detected at about 800 nm. The sensor plaster iselectrically connected to an electronic device comprising amicrocontroller and a battery, for data acquisition and their RFIDtransmission to an external computer (FIGS. 5 and 6).

Procedure.

SD rats are anesthetized with isoflurane, shaved on the back, then theelectronic near infrared device is attached. ABZWCY-HβCD is administered(5 mg/100 g, body weight) via intravenous injection. Transcutaneousmeasurement usually last 2 h.

Stock Solution Preparation.

An ABZWCY-HβCD/plasma stock solution is prepared by incubation of 500μg/ml ABZWCY-HβCD (in PBS solution) with rat plasma protein at 37° C.for 1 hour.

Plasma clearance kinetics for ABZWCY-HβCD is shown in FIG. 6: (a) 1exponential fitting (1e); (b) 3 exponential fitting (3e).

Example 4 Plasma Clearance Half-Life for ABZWCY-HβCD in the Presence ofProbenecid

In order to determine whether kidney tubular secretion had any effect onthe clearance of these markers, separate pharmacokinetic experimentsinvolving blockage of tubular secretion using Probenecid[p-(dipropyl-sulfamoyl)benzoic acid] were carried out.

The experimental protocol was approved and conducted in accordance withthe German Ministry of Health and according to the The National AnimalProtection Guidelines.

Animal:

female Sprague-Dawley rats.

Substances:

ABZWCY-HβCD.

Dosage:

ABZWCY-HβCD: 5 mg/100 g body weight; Probenecid: 50 mg/kg body weight.

Electronic Near Infrared Device for Transcutaneous FluorescenceDetection.

As described above.

Procedure:

SD rats are anesthetized with isoflurane, shaved on the back, then theelectronic near infrared detector device is attached. ABZWCY-HβCD isadministered (5 mg/100 g, body weight) via intravenous injection. Ratsare previously treated (30 min. before measurement) with Probenecid (50mg/kg body weight in 0.9% Saline, intraperitoneally). The transcutaneousmeasurement lasts approximately 2 h.

Plasma clearance kinetics for ABZWCY-HβCD in the presence of Probenecidis shown in FIG. 7: (a) 1 exponential fitting (1e); (b) 3 exponentialfitting (3e).

Plasma clearance half-life values measured in Examples 3 and 4 aresummarized in Table 2.

TABLE 2 Clearance half-life (minutes) Mean ± SD Conjugate 1-parameterfitting 3-parameter fitting ABZWCY-HβCD (n = 6) 17.7 ± 3.3 16.9 ± 4.7ABZWCY-HβCD + 18.5 ± 6.6 15.9 ± 5.5 Probenecid (n = 5)

1. A fluorescent compound of formula (I)F-L_(n)-CD_(n)  (I) wherein F is a tricarbocyanine residue of formula(II)

wherein R₁ and R₂ are independently selected from H, SO₃H, CO₂H, SO₂NH₂,CH₂COOH, NH₂, NHCOCH₂I, NO₂, Br, Cl, CH₃; R₃ and R₄ are independentlyselected from C₁₋₄ alkyl, (CH₂)₃C≡CH, (CH₂)₄C≡CH (CH₂)₅COOH, (CH₂)₃SO₃H,(CH₂)₄SO₃H, (CH₂)₃NH₂, (CH₂)₄NH₂, (CH₂)₃N⁺(CH₃)₃, (CH₂)₅N⁺(CH₃)₃,(CH₂)₃N₃, (CH₂)₄N₃, (CH₂)₃NHCOCH₂I, (CH₂)₄NHCOCH₂I; (CH₂CH₂O)₂CH₃,(CH₂CH₂O)₃CH₃ (CH₂CH₂O)₄CH₃; R₅, is H, Cl, or

wherein X is selected from NH, O, S; j is an integer from 1 to 4; k isan integer from 1 to 4; CD is a cyclodextrin residue of formula (III)

wherein m is and integer equal to 6, 7 or 8, R′, R″, R′″ areindependently selected from OH, OCH₃, OCH₂CH₃, OCH₂CHOHCH₃, OCHOHCH₃,OCH₂COOH, O(CH₂)₄SO₃H, N₃, NH₂, NHCOCH₃, OCH₂C≡CH, SH; L is a linkergroup resulting from the coupling of the tricarbocyanine of formula (II)to the cyclodextrin(s) of formula (III) according to the followingTable: Functional group of Functional group the tricarbocyanine of thecyclodextrin (F) in any (CD) in any of the groups of the groups R₃, R₄or R₅ R′, R″ or R′′′ Linker group (L) COOH OH —C(O)O— COOH NH₂ —C(O)NH—NCS OH —NC(S)O— NCS NH₂ —NC(S)NH— NH₂ COOH —NHC(O)— NHCOCH₂I SHNHC(O)CH₂S— C≡CH N₃

N₃ C≡CH

dichlorotriazine OH

dichlorotriazine OH, OH

dichlorotriazine OH, NH₂

n is an integer from 1 to 4, and salts thereof.
 2. A fluorescentcompound according to claim 1, wherein m is equal to 7 or
 8. 3. Afluorescent compound according to claim 1, wherein at least one of theR′, R″, R′″ groups is selected from OCH₃, OCH₂CH₃, OCH₂CHOHCH₃, whit theprovision that at least one group from R′, R″, R′″ is OH.
 4. Afluorescent compound according to claim 1, wherein R′ and R″ are OH, andR′″ is selected from OCH₃, OCH₂CH₃, OCH₂CHOHCH₃, preferably R′″ isOCH₂CHOHCH₃, wherein the substitution degree of R′″ is comprised between0.5 and 1.5 for each unit of Formula (III).
 5. A fluorescent compoundaccording to claim 1, wherein R₁ and R₂ are independently selected fromH, SO₃H and COOH.
 6. A fluorescent compound according to claim 1,wherein R₃ and R₄ are independently selected from methyl, ethyl,(CH₂)₅COOH, (CH₂)₄SO₃H, (CH₂)₃N⁺(CH₃)₃.
 7. A fluorescent compoundaccording to claim 1, wherein R₅ is selected from H, Cl, or


8. A fluorescent compound according to claim 1, wherein L is selectedfrom an ester, an ether, an amide or


9. A fluorescent compound according to claim 1, selected from: acompound of formula (IV):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of formula (I) isan ester bond formed in the coupling reaction of the carboxyl group ofthe tricarbocyanine with a residue group R′″═OH of CD; a compound offormula (V):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula is anester bond formed in the coupling reaction of the carboxyl group of thetricarbocyanine with a residue group R′″═OH of CD; a compound of formula(VI):

wherein CD is 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula is anester bond formed in the coupling reaction of the carboxyl group of thetricarbocyanine with a residue group R′″═OH of CD; a compound of formula(VII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ether bonds formed in the coupling reaction of adichlorotriazine group of the tricarbocyanine with a residue groupR′″═OH of CD₁ and CD₂, respectively; a compound of formula (VIII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (IX):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (X):

wherein CD₁, CD₂ and CD₃ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) are ester bonds formed in thecoupling reaction of a carboxyl group of the tricarbocyanine with aresidue group R′″═OH of CD₁, CD₂, and CD₃, respectively; a compound offormula (XI):

wherein CD₁, CD₂ and CD₃ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) are ester bonds formed in thecoupling reaction of a carboxyl group of the tricarbocyanine with aresidue group R′″═OH of CD₁, CD₂ and CD₃, respectively. a compound offormula (XII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (XIII):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (XIV):

wherein CD₁, CD₂, CD₃ and CD₄ are, independently,2-hydroxypropyl-β-cyclodextrin (HβCD) or 2-hydroxypropyl-γ-cyclodextrin(HγCD) and the linkers L of Formula (I) are ester bonds formed in thecoupling reaction of a carboxyl group of the tricarbocyanine with aresidue group R′″═OH of CD₁, CD₂, CD₃ and CD₄, respectively; a compoundof formula (XV):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (XVI):

wherein CD₁ and CD₂ are, independently, 2-hydroxypropyl-β-cyclodextrin(HβCD) or 2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linkers L ofFormula (I) are ester bonds formed in the coupling reaction of acarboxyl group of the tricarbocyanine with a residue group R′″═OH of CD₁and CD₂, respectively; a compound of formula (XVII):

wherein CD is a 2-hydroxypropyl-β-cyclodextrin (HβCD) or2-hydroxypropyl-γ-cyclodextrin (HγCD) and the linker L of Formula (I) isan ester bond formed in the coupling reaction of the carboxyl group ofthe tricarbocyanine with a residue group R′″═OH of CD.
 10. A fluorescentcompound according to claim 1 for use in the diagnosis of kidneyfunction, preferably in determining the glomerular filtration rate(GFR), in a mammal.
 11. A diagnostic formulation for use in thediagnosis of kidney function, comprising at least one fluorescentcompound according to claim
 1. 12. A method for diagnosing kidneyfunction of a mammal, comprising: (a) administering to the mammal atleast one fluorescent compound according to claim 1, and (b) detectingand measuring the fluorescence emission of the at least one fluorescentcompound.
 13. A method according to claim 12, wherein the administrationis intravenous.
 14. A method according to claim 12, wherein thefluorescence detection and measurement comprise the detection andmeasurement of fluorescence emerging from the mammalian skin in responseto excitation with a red light or near infrared light source.
 15. Themethod according to claim 12 wherein fluorescence is detected andmeasured by placing a sensor device onto the mammalian skin.
 16. Themethod according to claim 12, wherein the method is carried out over aclinically relevant measuring time.
 17. A screening method to identify atest agent suitable for the treatment of a chronic kidney disease in amammal comprising: i) administering to an animal model of chronic kidneydisease the test agent and at least one fluorescent compound accordingto claim 1, wherein the at least one fluorescent compound isadministered subsequently to the test agent; ii) measuring theglomerular filtration rate (GFR) of the animal model by detecting andmeasuring the fluorescence emission of the at least one fluorescentcompound, wherein the detection and measurement of the fluorescencecomprises the detection and measurement of the fluorescent emissionemerging from the skin of the animal model in response to excitationwith a red light or near infrared light source; iii) selecting the testagent that increases the glomerular filtration rate.